Endothelial lipase variant T111I does not alter inhibition by angiopoietin-like proteins

High levels of HDL-C are correlated with a decreased risk of cardiovascular disease. HDL-C levels are modulated in part by the secreted phospholipase, endothelial lipase (EL), which hydrolyzes the phospholipids of HDL and decreases circulating HDL-C concentrations. A 584C/T polymorphism in LIPG, the gene which encodes EL, was first identified in individuals with increased HDL levels. This polymorphism results in a T111I point mutation the EL protein. The association between this variant, HDL levels, and the risk of coronary artery disease (CAD) in humans has been extensively studied, but the findings have been inconsistent. In this study, we took a biochemical approach, investigating how the T111I variant affected EL activity, structure, and stability. Moreover, we tested whether the T111I variant altered the inhibition of phospholipase activity by angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), two known EL inhibitors. We found that neither the stability nor enzymatic activity of EL was altered by the T111I variant. Moreover, we found no difference between wild-type and T111I EL in their ability to be inhibited by ANGPTL proteins. These data suggest that any effect this variant may have on HDL-C levels or cardiovascular disease are not mediated through alterations in these functions.

www.nature.com/scientificreports/In this study, we evaluated the EL variant with a T111I substitution for phospholipase activity, stability, and ability to be inhibited by ANGPTL proteins.
To address the potential effect of the T111I mutation on EL function, we generated expression constructs coding for wild-type and T111I mutant EL (Fig. 1a).When these constructs were transiently expressed in HEK 293 T cells, we found that expression, secretion, and cleavage of the EL T111I mutant was similar to wild-type EL (Fig. 1b).Consistent with a previous report 23 , we also found that phospholipase activity of the T111I mutant was similar to wild-type, whether using an artificial substrate (Fig. 1c), or when measuring hydrolysis of phospholipids of human HDL (Fig. 1d).

Stability and structure of EL T111I
The structure of EL has not been solved.We utilized ColabFold 33 , a derivative of Alphafold2 34 , to predict the structure of wild-type human EL (Fig. 2a).In agreement with previously published predictions 35 , the T111 residue is located at the end of an alpha helix in the N-terminus domain of EL.In tandem with the predicted structure, we utilized Dynamut2 36 to predict how the single mutation, T111I, might alter the stability of EL.Using Dynamut2 we calculated the ΔΔG of the T111I mutation using the top 5 predicted EL structures as outputted by ColabFold.The predicted ΔΔG Stability was − 0.18 kcal/mol for the top ranked model (the model shown in Fig. 2a).The range for all 5 models was − 0.39 kcal/mol to − 0.14 kcal/mol.When we used Dynamut2 to calculate the ΔΔG Stability of WT EL as determined by AlphaFold2 (AF-Q9Y5X9-F1), the ΔΔG Stability was − 0.33 kcal/mol.These values indicate that the T111I mutation could potentially be mildly destabilizing 37 .We have previously observed that wild-type EL spontaneously loses activity at 37 °C38 , a trait shared with the closely related lipase, lipoprotein lipase 39,40 .Therefore, we asked if the T111I EL spontaneously lost activity at a different rate than wild-type EL.However, we found that, at 37 °C, EL with the T111I mutation lost activity at a similar rate compared to wild-type (Fig. 2b).The loss of activity for both WT and T111I EL did not appear to be mediated by proteolysis as there was no evidence of novel cleavage or increased degradation over time in these samples (Fig. 2c).
Because EL is closely related to LPL, we asked if the T111 mapped to an area homologous to an ANGPTL4 binding domain in LPL.We aligned the protein sequence of human EL against human LPL using the Clustal format alignment by MAFFT 50 (Fig. 3a).When comparing homology between EL and LPL, the T111 residue www.nature.com/scientificreports/aligns with one of the binding site clusters to which ANGPTL4 binds (Fig. 3a,b).Therefore, it is possible that ANGPTL4 and/or ANGPTL3 interacts with EL at the same site, and that the T111I mutation could disrupt this interaction.To test this idea, we first measured EL phospholipase activity after incubation with increasing concentrations of human or mouse ANGPTL3 protein.Inhibition of both wildtype and T111I EL was dose-dependent, consistent with our previous findings 38 .Moreover, both human and mouse ANGPTL3 inhibited T111I EL to a similar level as wild-type EL (Fig. 4a,b).Recently ANGPTL4 has also been reported to inhibit EL 51 .Therefore, we asked if ANGPTL4 inhibition was affected by the T111I mutation.As with ANGPTL3, there was little difference in inhibition of T111I EL by ANGPTL4 when compared to wild-type EL (Fig. 4c,d).Although we previously showed that ANGPTL8 did not affect the ability of ANGPTL3 to inhibit EL 38 , we considered the possibility that ANGPTL8 would have an effect on T111I EL.However, we saw little difference in the inhibition of T111I EL when comparing ANGPTL3 and ANGPTL3 with ANGPTL8 (Fig. 4e).
In vivo, EL can be bound to the surface of endothelial cells through interactions with heparan sulfate proteoglycans (HSPGs) 52 , and we have previously found that EL bound to endothelial cells is resistant to ANGPTL3 inhibition 38 .We therefore asked if the T111I substitution affected the ability of endothelial cell-bound EL to be inhibited by ANGPTL3 or ANGPTL4.We stably expressed WT EL or T111I in rat heart microvessel endothelial cells (RHMVECs) (Fig. 5a).To evaluate if the EL protein produced by these cell lines was properly secreted and bound to the outside of the cell, we treated EL expressing cells with 10 U/mL heparin to release HSPG-bound EL from the membrane.We found that the majority of both WT and T111I EL was released into the media after heparin treatment, suggesting appropriate membrane localization (Fig. 5b).To test inhibition of membranebound EL, we treated WT and T111I EL expressing RHMVECs with ANGPTL3 or ANGPTL4 and measured phospholipase activity.As expected, EL bound to the endothelial cell surface was resistant to ANGPTL3 and ANGPTL4 inhibition (Fig. 5).However, we again found little difference between WT and T111I EL as far as inhibition by ANGPTL3 (Fig. 5c,d) or ANGPTL4 (Fig. 5e,f).

Discussion
In this study, we evaluated a common EL variant, T111I, for its effects on EL activity, stability, and ability to be inhibited by ANGPTL proteins.In all these parameters, we found that EL T111I was no different than wildtype EL.
The 584C/T polymorphism, which encodes the T111I mutation in EL, has been associated with increased HDL levels in some population studies.The variant was originally discovered by sequencing black and Caucasian individuals with high HDL-C levels 11 .Subsequently, several groups found an association between the T111I variant and HDL-C levels and other cardiovascular risk factors 10,[12][13][14][15][16][17] , but several other studies found no association [18][19][20][21][22][23] .Numerous studies continue to be performed to evaluate a potential association between this EL variant, HDL-C levels, and the risk of CAD.A meta-analysis, performed by Cai et al. in 2014, included 9 case-control studies of Caucasian and Asian populations.This meta-analysis, which considered the sample size of the previously published studies and repeated the statistical analysis, found that carriers of the 584C/T polymorphism did have higher HDL-C levels compared to non-carriers but did not find a significant association between the variant and the risk of coronary heart disease 17 .A second meta-analysis performed by Zhao et al. in 2020 included 13 studies from countries including The United States, China, Japan, Netherlands, Iran, Egypt, and Turkey.This analysis did not examine association with HDL-C levels, but did find that EL 584C/T www.nature.com/scientificreports/ was associated with CAD susceptibility 53 .A third meta-analysis by Wu et al. included 14 case-control studies with 9731 subjects 54 .This meta-analysis, which again did not analyze HDL-C levels, also found a significant association between the EL 584C/T polymorphism and CAD, but only in African populations 54 .It is important to note, however, that the authors of this study clearly disclosed that only one of the fourteen eligible studies could be analyzed for the African populations.Thus, although an association may be possible between T111I and HDL-C or CAD for different ethnic groups, the lack of eligible studies may influence the ability to confidently draw conclusions.Moreover, even if the T111I allele is associated with increased cardiovascular risk, it may not be causal, with causality coming from other alleles in the same haplotype 22,35 .In summary, the association between the EL T111I variant and HDL-C or the risk of CAD remains somewhat unsettled, and there is, to date, no evidence of causality.If T111I EL is indeed associated with high HDL levels, the simplest explanation is that it alters EL function.Functional changes could include changes in enzymatic activity, changes in stability, and changes in interactions with other proteins, such as the endogenous inhibitor ANGPTL3.A previous study by Edmonson et al. found no difference in phospholipase activity between wild-type and T111I EL when tested either by measuring the hydrolysis of dipalmitoylphosphatidyl choline or by using isolated human HDL 3 as a substrate 23 .We likewise found no differences in phospholipase activity using either a fluorescence-based assay or fatty-acid release from human HDL particles.
Although the structure of EL has not been solved experimentally, the ColabFold structure puts the T111 at the end of an alpha helix in the N-terminal domain of EL.Although the N-terminal domain contains the catalytic domain, T111 is not near the active site, nor does it seem positioned to contribute to proper folding of EL.We found no evidence that the T111I mutation disrupts EL stability, at least in vitro.These data support previous predictions which utilized homology modelling to predict the structure of EL and CHARMM calculation protocols to predict stability changes and suggested that the T111I mutation does not have any significant effects on the EL protein stability 35 .
The location of T111 on the outer face of an alpha helix on EL suggests that it could potentially be involved in interactions with other proteins.Indeed, in the homologous lipase, LPL, this same alpha helix is thought to interact with the lipase inhibitor ANGPTL4 39,49 .However, it is important to note that T111 is not conserved between EL and LPL.Moreover, we did not observe any meaningful differences in inhibition by either ANGPTL3 or ANGPTL4 when threonine 111 was mutated to isoleucine.We conclude that if T111 is involved in protein binding, it is not with ANGPTL proteins.
The data in this paper suggest that the T111I variation does not alter EL specific activity, protein stability, or inhibition by ANGPTL proteins.However, it is important to note that it is also possible that T111I induces small enough changes in EL activity, stability, or inhibition by ANGPTL proteins that the sensitivity and reproducibility of our assays were unable to detect these differences.Moreover, our findings do not rule out the possibility that T111I alters or compromises EL function in other ways that could potentially alter HDL levels or function.The T111I variant introduces a large hydrophobic residue on the surface of EL, which would increase the propensity for non-specific hydrophobic associations or could alter the interactions of EL with proteins other than ANGPTLs.This change in hydrophobicity could also change the identity and specificity of lipoprotein binding to EL. T111I EL might bind preferentially to different sizes of HDL compared to wildtype EL or bind more strongly to non-HDL lipoproteins thus interfering with HDL binding.Such changes could also alter the bridging function of EL 52,55 .Subtle changes in EL structure cause by the T111I variant could also alter post-translational modifications or substrate specificity.Any of these changes could alter EL function in vivo.An alteration in substrate specificity, for example, could result in changes in HDL lipid composition and subsequent changes in the proteome and functionality of HDL.These possibilities could be explored by using mass spectrometry to examine the lipid and protein composition of HDL after incubation with wildtype or T111I EL.HDL functions, including cholesterol efflux, paraoxonase activity, and anti-inflammation could also be assayed following EL incubation.It is unclear, however, given the uncertainty of the connection between T111I and any physiological changes, how much continued effort should be put into pursuing possible differences between wild-type and T111I EL.

Production and quantification of ANGPTL3, ANGPTL4, and ANGPTL3 + 8 conditioned media
To produce human and mouse ANGPTL3 and ANGPTL4 protein, HEK 293 T cells were grown to 80% confluency in T25 flasks in complete DMEM.Cells were transfected with 5 μg of pHS18 (strep-tagged mouse ANGPTL3), pHS15 (strep-tagged human ANGPTL3), pHS5 (V5-tagged mouse ANGPTL4), or pHS2 (V5-tagged human ANGPTL4) and 10 μl of 1 mg/ml PEI (polyethylenimine).To produce mouse ANGPTL3 and ANGPTL8 complexes, HEK 293 T were grown to 80% confluency in T75 flasks and transfected with 5 μg of pHS18 and 5 μg of pWL1 (V5-tagged mouse ANGPTL8) simultaneously.Cells were switched to serum-free DMEM and 1X ProteaseArrest protease inhibitor cocktail (APExBIO) 24 h post-transfection.Conditioned media were collected 48-72 h later.To produce 293 T control media (CM), HEK293T cells were transfected and media collected in the same manner, only no DNA was added to the transfection.This control conditioned media was used as a control for all experiments involving ANGPTL conditioned media.The concentration of ANGPTL3 in conditioned media was determined via western quantification against a batch of mouse ANGPTL3 with a known concentration, previously quantified via ELISA kit (RayBiotech, catalog #ELM-ANGPTL3-1).The concentration of ANGPTL4 in conditioned media was determined via western quantification against a batch of human ANGPTL4 with a known concentration, previously quantified via a human ANGPTL4 ELISA kit (Sigma).

Production and quantification of endothelial lipase conditioned media
To produce EL conditioned media, HEK 293 T were grown to 80% confluency in DMEM supplemented with 5% FBS, Penicillin/streptomycin antibiotics, and L-Glutamine.Cells were transfected with 10 μg of pKS6 (human WT EL) or pKS17 (human T111I EL) and 20 μl of 1 mg/ml PEI (polyethylenimine).Media was changed to serumfree OptiMEM containing 1X ProteaseArrest (APExBIO) and 0.1 U/ml heparin (Fresenius Kabi USA, LLC) 24 h post-transfection.EL conditioned media was collected after an additional 24 h.Protein expression was confirmed by western blotting and tested via phospholipase activity assay.EL concentration was determined by comparing EL protein signal with a quantified batch of strep-tagged human EL protein via quantitative western blot.

Western blot
Protein samples were size fractionated on 12% SDS-PAGE gels and then transferred to a nitrocellulose membrane.Membranes were blocked with casein buffer (1% casein, Fisher Science Education).Primary antibodies were diluted in casein buffer + 0.1% Tween.Primary antibody dilutions were 1:3000 for a mouse monoclonal antibody against EL (LIPG antibody clone 3C7, Lifespan Biosciences), 1:2000 for a goat antibody against beta-actin (Abcam), 1:3000 for a rabbit polyclonal antibody against Strep-tag II (Abcam) to detect Strep-tagged ANGPTL3, or 1:6000 for a mouse monoclonal antibody against V5 tag (R960-25; Invitrogen) to detect V5-tagged ANGPTL4.After washing with PBS-T, membranes were incubated with Dylight680-or Dylight800-labeled secondary antibodies (Thermo Scientific) diluted 1:5000 in casein.After washing with PBS-T, antibody binding was detected using an Odyssey Clx Infrared Scanner (Li-Cor).

EL activity assays
Phospholipase activity was monitored using the EnzChek Phospholipase A1 assay kit (ThermoFisher) as described previously 58 .EL and ANGPTL3 or ANGPTL4 proteins in conditioned media were combined and incubated at 37 °C for 30 min.Following incubation, 50 μl of sample was mixed with 50 μl of substrate solution and were incubated at room temperature (approximately 20-22 °C) for 30 min, reading fluorescence (485 nm excitation/515 nm emission) every 1 or 2 min with an Infinite F200 plate reader (Tecan).Relative phospholipase activity was calculated by calculating the slope of the linear part of the curve (typically in the range between 5 to 25 min) and then subtracting out the slope of the blank (sample with no EL conditioned media).
To detect phospholipase activity on the cell surface, RHMVECs expressing WT EL (pKS7-RHMVEC) or T111I EL (pKS18-RHMVEC) were grown to confluency in 96-well clear-bottom plates coated with fibronectin.

Figure 1 .
Figure 1.Expression and activity of the EL T111I mutation.(a) Sequencing data showing successful generation of the 584C/T mutation in human LIPG.(b) Expression and secretion of wild-type and T111I EL.Western blot shows expression of EL in the lysate (lys) and media of 293 T cells transfected with wild-type or T111I EL.Uncropped blot shown in Supplemental Fig. 1a.(c) Activity of three independently collected batches of WT and T111I EL.For each batch constructs expressing WT and T111I EL were transfected at the same time.After matching protein levels via western blot intensity, phospholipase activity of the conditioned media was measured using a fluorescence-based phospholipase activity assay.Bars show mean ± SD.P values determined using student t test.(d) Phospholipase activity of EL as measured by the release of NEFAs from HDL.The indicated concentrations of EL were combined with human HDL for 60 min at 37 °C.Points represents 3 independent experiments (mean ± SD).P value determined by one phase decay least squares fit.

Figure 2 .
Figure 2. Stability of EL T111I.(a) Structure of WT EL as predicted by ColabFold.The catalytic triad of EL is depicted in green and the residue T111 is colored in red.(b) Activity over time of WT and T111I EL conditioned media incubated at 37 °C.Activity at each time point was measured using a fluorescence-based phospholipase activity assay.Points represents 5 independent experiments (mean ± SD).P value determined by one phase decay least squares fit.(c) Western of T111I and wild-type EL after incubation at 37 °C with increasing time.Uncropped blot shown in Supplemental Fig. 1a.

Figure 3 .
Figure 3. Potential ANGPTL binding sites on EL.(a) MAFFT multiple sequence alignment of human LPL and human EL.ANGPTL4 binding sites on LPL and the homologous regions of EL are boxed in purple.The T111 residue is boxed in red.(b) Predicted structure of WT EL indicating the catalytic triad (green) and the regions homologous to the ANGPTL4 binding sites on LPL (purple).

Figure 4 .
Figure 4. Inhibition of EL T111I by ANGPTL3 and ANGPTL4.WT EL and T111I EL were incubated at 37 °C for 30 min with increasing concentrations of (a) mouse ANGPTL3, (b) human ANGPTL3, (c) mouse ANGPTL4, (d) human ANGPTL4, or (e) mouse ANGPTL3-ANGPTL8 complexes.Activity was measured using a fluorescence-based phospholipase activity assay.Points represent mean (± SD) of 4 experiments each run with 2 biological duplicates.P values determined by one phase decay least squares fit.

Figure 5 .
Figure 5. Inhibition of membrane-tethered EL T111I by ANGPTL3 and ANGPTL4.(a) Expression of WT and T111I EL from transduced RHMVEC.Western blot shows expression of EL in the lysate of virally transduced endothelial cells.(b) Release of HSPG-bound wild-type and T111I EL from the cell membrane.Western blot shows EL in lysate and media of wild-type and T111I expressing cells treated with ± 10 U/mL heparin for 24 h.Uncropped blots shown in Supplemental Fig. 1b,c.(c-f) WT EL and T111I EL expressing RHMVEC were incubated at 37 °C for 30 min with increasing concentrations of (c) mouse ANGPTL3, (d) human ANGPTL3, (e) mouse ANGPTL4, or (f) human ANGPTL4.Activity was measured using a fluorescencebased phospholipase activity assay.Points represent mean (± SD) of 4 experiments each run with 2 biological duplicates.P values determined by one phase decay least squares fit.
was then appended to the C terminus of the open reading frame using Phusion site-directed mutagenesis (New England Biolabs).